Skp2 is required for survival of aberrantly proliferating Rb1-deficient cells and for tumorigenesis in Rb1+/- mice

Abstract

Heterozygosity of the retinoblastoma gene Rb1 elicits tumorigenesis in susceptible tissues following spontaneous loss of the remaining functional allele. Inactivation of previously studied retinoblastoma protein (pRb) targets partially inhibited tumorigenesis in Rb1(+/-) mice. Here we report that inactivation of pRb target Skp2 (refs. 7,8) completely prevents spontaneous tumorigenesis in Rb1(+/-) mice. Targeted Rb1 deletion in melanotrophs ablates the entire pituitary intermediate lobe when Skp2 is inactivated. Skp2 inactivation does not inhibit aberrant proliferation of Rb1-deleted melanotrophs but induces their apoptotic death. Eliminating p27 phosphorylation on T187 in p27T187A knock-in mice reproduces the effects of Skp2 knockout, identifying p27 ubiquitination by SCF(Skp2) ubiquitin ligase as the underlying mechanism for Skp2's essential tumorigenic role in this setting. RB1-deficient human retinoblastoma cells also undergo apoptosis after Skp2 knockdown; and ectopic expression of p27, especially the p27T187A mutant, induces apoptosis. These results reveal that Skp2 becomes an essential survival gene when susceptible cells incur Rb1 deficiency.

Fig. 1

Roles of Skp2 in spontaneous tumorigenesis in Rb1^+/- mice and in ENU-induced tumorigenesis. a. Expression of the indicated proteins in wild type normal pituitary glands and pituitary tumors developed in Rb1^+/-Skp2^+/+ mice, determined by Western blot. b. Levels of Skp2 mRNA in pituitary glands and pituitary tumors (developed in Rb1^+/- mice), determined by Q-PCR normalized with GAPDH. c. Incidence for pituitary and thyroid tumors at various stages in Rb1^+/-Skp2^+/+ and Rb1^-/-Skp2^-/- mice. p values are by Fisher’s exact tests (various lesions were combined for analyses). d. Kaplan-Meier survival analysis for the indicated mice. p value is by Log Rank test. One Rb1^+/-Skp2^-/- mouse died at thirteen months and one died at sixteen months with macroscopically normal pituitary and thyroid glands. The causes of death were unclear with a possible association with eye and skin lesions. e. Kaplan-Meier survival analysis for the indicated mice treated with ENU.

Fig. 2

Effects of targeted deletion of Rb1 in pituitary IL and AL of Skp2^+/+ and Skp2^-/- mice. a. The POMC-Cre strain induced Cre-loxP-mediated excision in PL and AL of Skp2^+/+ and Skp2^-/- mice. Rosa26R, Rosa26-loxP-STOP-loxP-EYFP. Mice were examined at 4 weeks of age. EYFP expression was by fluorescence of frozen-sectioned samples. b. Pituitary ILs of indicated mice at 7 weeks of ages. H&E stained sections of various pituitaries are shown. Big insert is enlarged view of areas marked by the small box. c. Pituitary glands of the indicated mice at the indicated ages, examined as in panel a. Scale bar, 200 μm.

Fig. 3

Effects of Skp2 inactivation on E2F deregulation, aberrant proliferation and apoptosis, and p27 expression in pituitary ILs following Rb1 deletion. Various indicated mice at the ages of PND 10 (post-natal day 10) and 4 weeks are presented in panels a and b, respectively. E2F deregulation is examined by PCNA expression, proliferation by Ki67 expression, and apoptosis by TUNEL labeling. Quantification of Ki67 and TUNEL labeling in ILs was performed with three pituitaries of each indicated genotypes at the indicated ages. Rb1 genotypes indicate the outcome of Cre-loxP mediated deletion in IL. p values are by t test. Error bars are s.d. Scale bar, 200 μm.

Fig. 4

Effects of targeted deletion of Rb1 in pituitary IL of p27T187A KI mice. a. IL morphology, PCNA expression, Ki67 and TUNEL labeling, and p27 expression were examined at the indicated ages, with quantification of Ki67 and TUNEL labeling presented in b and c, respectively. p values are by t test. Error bars are s.d. d. IL morphology and PCNA expression after Rb1 deletion in p27^T187A/+ mice at 7 and 11 weeks of age. Scale bar, 200 μm.

Fig. 5

Effects of Skp2 knockdown and stabilized p27 expression on established Y79 cells and early passage RB177 retinoblastoma cells. (a-e) Y79 and RB177 cells infected with lentiviruses expressing shRNA targeting Skp2. Two independent Skp2 shRNAs and a scrambled shRNA control (Scrm) were used as indicated. After drug selection, infected cells were evaluated for Skp2 mRNA by quantitative RT-PCR (a), cell proliferation by counting live cells (b), cell cycle profile by FACS (c), apoptosis by TUNEL staining (d), and p27 expression by Western immunoblotting, with Cdk2 as a loading control (e). (f-i) Y79 and RB177 cells infected with BE-GFP lentiviral vector encoding p27 or p27T187A. Infected cells were evaluated for p27 expression (f), cell proliferation (g), cell cycle profile (h), and TUNEL staining (i), (j-k) Y79 cells transduced with BE-GFP vector or BE-GFP-RB, followed 2 days later by transduction with Skp2 shRNA or scrambled shRNA control (j) or with BE-GFP or BE-GFP-p27T187A (k), and evaluated cells with sub-G1 DNA content. Averages with s.d. are shown. Asterisks indicate P < 0.05 relative to applicable controls. l. A new model of tumorigenesis after Rb1 loss. Two consecutive arrows suggest the presence of multiple steps between them.